**1. Introduction**

To date, although testicular spermatozoa from patients with non-obstructive azoospermia (NOA) have been used widely for intracytoplasmic sperm injection (ICSI), this method's effectiveness still has potential for further improvement. NOA is characterized by the absence of any spermatozoa, whether dead or alive, in the ejaculate due to reduced or nonexistent sperm production in the testicle [1]. Testicular fine needle aspiration (FNA or testicular sperm aspiration—TESA) is an effective and non-invasive method used to obtain sperm, especially from patients with obstructive azoospermia [2]. Although its simpler and less traumatic features have made FNA the preferred method, testicular sperm extraction (TESE) is the treatment of choice for patients with NOA, with a satisfactory number of successful spermatozoa retrieved in approximately half of the patients. In the conventional TESE procedure, the testis is exposed through a small incision in the tunica albuginea, and multiple biopsies are taken randomly [3]. However, microTESE carried out at high magnification under an operating microscope allows visualization of whitish, larger, and more opaque seminiferous tubules likely to contain mature germ cells [4]. Although not randomized, most studies have reported that the sperm recovery rate from microTESE is superior to that from conventional TESE [5–7]. In fact, a recent controlled, randomized study verified the efficacy of microTESE compared with that of TESE in retrieving spermatozoa from patients with NOA [8]. In addition to the surgical technique, however, in vitro extraction of sperm from surgically excised testicular tissue or tubules is also important for obtaining spermatozoa of sufficient quality and quantity for use in ICSI.

**Citation:** Aydos, K.; Aydos, O.S. Sperm Selection Procedures for Optimizing the Outcome of ICSI in Patients with NOA. *J. Clin. Med.* **2021**, *10*, 2687. https://doi.org/10.3390/ jcm10122687

Academic Editors: Giovanni M. Colpi and Ettore Caroppo

Received: 24 May 2021 Accepted: 16 June 2021 Published: 18 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **2. Processing and Selection of Surgically Retrieved Sperm for ICSI**

*2.1. Mechanical Processing of Testicular Tissue*

The goal of TESE treatment in patients with NOA is to retrieve spermatozoa suitable for ICSI from the testicular tissue obtained by surgical intervention. Different methods have been described for processing TESE specimens in the laboratory. The most preferred tissue-processing procedure is mechanical treatment of testicular tissue pieces by shredding and mincing with fine needles, scissors, or glass slides [9–11]. In addition to the shredding, tubule pieces cut into short lengths can be squeezed into the medium with the help of a bent pipette [12,13]. The suspension can then be processed using either the swim-up or density gradient centrifugation method. In the former, following the sedimentation of tissue fragments, the most motile spermatozoa swim up the medium; in the density gradient method, during centrifugation, sperm cells are separated according to swimming rate while moving through discrete layers of density gradients [14,15]. Both methods have their advantages. Verheyen et al. compared rough shredding, fine mincing, vortexing, and crushing methods to evaluate the efficiency of obtaining a maximum number of sperm from testicular biopsy specimens, and fine mincing of testicular tissue followed by discontinuous Percoll centrifugation was found to be the most effective method of isolating a pure fraction of spermatozoa available for ICSI as well as cryopreservation [16]. However, the risk of cell loss through the discrete layers in Percoll gradient separation cannot be ignored. Therefore, if too few cells are present, it may be preferable to carefully mince the testicular tissue with fine forceps or microscissors and centrifuge the entire suspension immediately [10,17]. As another option, Haimov-Kochman et al. suggested that leaving tissue fragments from TESE in medium for 10 min and then centrifuging the supernatant could yield rapid sperm recovery without wasting time shredding the tissue [18]. Interestingly, when the supernatant was spermatozoa-negative, no spermatozoa were present in the tissue either. Therefore, this method makes it possible to quickly predict the success of TESE. Nevertheless, when it comes to choosing the optimal method, in addition to the structural features of the testicular tissue, personal experience and laboratory facilities should also be taken into consideration.

#### *2.2. Use of Erythrocyte-Lysing Buffer (ELB)*

One of the most frequently encountered problems during the search for spermatozoa in fragmented TESE specimens is abundant erythrocyte infiltration. Attempting to identify rare spermatozoa among dense erythrocyte clusters is time consuming and associated with a reduced recovery rate. The use of erythrocyte lysing buffer (ELB) for the elimination of red blood cells present in the mechanically shredded testicular tissues of patients with NOA was first described by Verheyen et al. [19]. Resuspending the testicular sperm pellet in ELB (155 mM NH4Cl, 10 mM KHCO3, and 2 mM EDTA; pH 7.2) yielded recovery of additional motile spermatozoa in a shorter time. Treatment of TESE samples with ELB shortens the processing time and increases the success of cell retrieval without decreasing the fertilization potential of the embryo [17]. However, treatment of sperm suspensions with ELB has also been shown to impair sperm quality [20]. When spermatozoa were incubated in ELB for 10 min, their motility and viability decreased and DNA fragmentation increased. This effect of ELB on sperm may be due to the fact that the same mechanism of disruption in ammonium equilibrium responsible for the lysis in erythrocytes is also present in the sperm. In addition, osmotic stress caused by the chemical structure of this buffer may damage the plasma membrane and alter sperm metabolism [21]. Conversely, Soygur et al. showed that ELB itself and the cellular stress caused by erythrocyte lysis did not have detrimental effects on the survival of ejaculated sperm [22]. According to their protocol, however, ELB incubation was only allowed for 5–10 s, so there may not have been enough time for damage to occur. Despite the fact that the potential influence of this buffer on sperm parameters remains uncertain, at present, ELB medium is widely used during testicular germ cell extraction to clean the cellular suspension from erythrocytes that interfere with visualization [23,24].
